Resorbable crosslinked form stable membrane for use outside the oral cavity

ABSTRACT

The invention relates to resorbable crosslinked form stable membrane which comprises a composite layer of collagen material and inorganic ceramic particles containing 1.5 to 3.5 weight parts of inorganic ceramic for 1 weight part of collagen material, sandwiched between two layers of elastic pretensed collagen material (collagen material that has been stretched such as to be in the linear/elastic region of the stress-strain curve), the collagen material comprising 50-100% (w/w) collagen and 0-50% (w/w) elastin, and has shape and dimensions suitable for use in human tissue regeneration outside the oral cavity in rhinoplasty, postlateral spinal fusion or orbital reconstruction.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of European Patent Application No.17174189 filed on Jun. 2, 2017, the disclosure of which is incorporatedherein in its entirety by reference.

FIELD OF THE INVENTION

The invention relates to a resorbable crosslinked form stablecomposition for use in human tissue regeneration outside the oralcavity, notably in the craniofacial region and the cervical, thoracic,lumbar or sacral region of the spine, or in craniotomy reconstructivesurgeries.

BACKGROUND OF THE INVENTION

In order to regenerate non-containing bony defects by bone formation,such as e.g. in horizontal or vertical augmentations in the maxilla ormandible, mechanical stabilization of the defect is required (Bendkowski2005 Space to Grow in The Dentist, Merli et al. 2007 Vertical ridgeaugmentation with autogenous bone grafts: resorbable barriers supportedby ostheosynthesis plates versus titanium-reinforced barriers. Apreliminary report of a blinded, randomized controlled clinical trial inInt J Oral Maxillofac Implants, Burger 2010 Use of ultrasound-activatedresorbable poly-D-L-lactide pins (SonicPins) and foil panels (Resorb-X)for horizontal bone augmentation of the maxillary and mandibularalveolar ridges in J Oral Maxillofac Surg and Louis 2010 Vertical ridgeaugmentation using titanium mesh in Oral Maxillofac Surg Clin North Am).Indeed, oral tissues are exposed to complex mechanical forces duringmastication, swallowing, tongue movement, speech, tooth movement andorthodontic treatment. Especially during wound healing followingsurgical procedures, internal and external forces may occur, creatingpressure, shear forces and bending moments upon the regenerative deviceand newly formed tissue.

A form stable membrane resisting those forces is a useful means forbringing that mechanical stabilization.

It is known to use for that purpose Ti-meshes, Ti-plates orTi-reinforced PTFE form stable membranes which have to be removed afterbone regeneration during a second surgery. An example of a commerciallyavailable Ti-reinforced form stable membrane is the Cytoplast® membranemarketed by Osteogenics. However, the occurrence of dehiscences or othercomplications when using expanded Ti-reinforced membranes is reported tobe high (Strietzel 2001 Risks and complications of membrane—guided boneregeneration. Retrospective analysis in Mund Kiefer Gesichtschir, Merliet al. 2007 see above and Rocchietta et al. 2008 Clinical outcomes ofvertical bone augmentation to enable dental implant placement: asystematic review in J Clin Periodontol).

Non reinforced PTFE membranes were widely used prior the introduction ofresorbable collagen membranes in 1996, but disappeared very fast afterthe introduction of collagen membranes.

To avoid the need of removal of a form stable membrane or meshes in asecond surgery, a resorbable form stable membrane is of interest.Several resorbable form stable membranes or meshes have been described,essentially made from PLA (poly-lactic acid) or PLGA(poly-lactic-co-glycolic acid). Examples are notably (1) “Sonic WeldRX®” and “Resorb-X®” from KLS Martin, (2) “Guidor®” from SunstarAmericas, (3) the “Inion GTR System™” from Curasan and (4) “RapidSorb®”from Depuy Synthes. The disadvantage of those membranes is that duringtheir in vivo hydrolytic degradation they release lactic and/or glycolicacid which cause tissue irritation and histological signs of a disturbedwound healing (Coonts et al. 1998 Biodegradation and biocompatibility ofa guided tissue regeneration barrier membrane formed from a liquidpolymer material in J Biomed Mater Res, Heinze 2004 A space-maintainingresorbable membrane for guided tissue regeneration in Business breifing:Global Surgery and Pilling et al. 2007 An experimental in vivo analysisof the resorption to ultrasound activated pins (Sonic weld) and standardbiodegradable screws (ResorbX) in sheep in Br J Oral Maxillofac Surg).

To overcome PLGA/PLA associated wound healing problems, the use ofautologous bone blocks from the patient and partly or completelypurified bone blocks, such as e.g. Geistlich Bio-Oss® Block (GeistlichPharma A.G.) or Puros® Allograft Block (RTI Surgical Inc.), is widelyaccepted. Autologous bone blocks have the disadvantage that they areharvested from a second site leading to more pain. (Esposito et al. 2009The efficacy of horizontal and vertical bone augmentation procedures fordental implants—a Cochrane systematic review in Eur J Oral Implantol).

To enable the use of autologous bone chips harvested during surgery,usually in combination with xenogenic bone graft particles, the socalled bone shield technique was developed using autologous corticalbone from the mandibula (Khoury et al. 2007 Bone Augmentation in OralImplantology Quintessence). Disadvantages of this procedure are that itis extremely technique sensitive and that it is associated with secondsite morbidity and more pain. Further, bone shields are applied onlylaterally, therefore no mechanical protection is given from the coronalaspect of the defect. The term “bone shield” was used for advertisingPLA/PLGA membranes as well as a partially demineralized cortical boneshield (Semi-Soft and Soft Lamina Osteobiol® from Tecnoss). Thedisadvantages of this demineralized bone shield are that bent boneshields have to be fixed always, that they are relatively thick comparedto e.g. Ti-reinforced PTFE membranes and that they come only in roundshapes with curved edges on the coronal aspect of the bony defect. Fordentists, a 6-8 mm wide plateau in the coronal aspect of the ridge wouldbe much more preferred (Wang et al. 2002 HVC ridge deficiencyclassification: a therapeutically oriented classification in Int JPeriodontics Restorative Dent).

An attempt to combine uneventful healing and form stability is theresorbable form stable collagen membrane disclosed in U.S. Pat. No.8,353,967-B2, which is prepared from a collagen suspension in 5-25%ethanol/water in a mould by freeze-drying and heating at 100 to 140° C.Such a membrane is manufactured by Osseous Technologies of America andmarketed under the trade name “Zimmer CurV Preshaped Collagen Membrane”by Zimmer. However, that commercial membrane has weak form stability anda thickness of about 1.5 mm rising after incubation in saline to aroundabout 2.3 mm; this may lead to a risk of a high dehiscence rate.

In summary the current solutions are thus not fully satisfying fordentists or patients. Either a second surgery is necessary and/or thereis a high risk of eventful wound healing. Solutions which are notassociated with a high risk of eventful wound healing are either notform stable membranes, require a second surgery or have otherdisadvantages.

US 2013/0197662 discloses a process for fabricating a biomaterialcomprising a) joining a porous collagen-based material with a non-porouscollagen-based material by applying a controlled amount of a gelcomprising collagen to a bonding surface of the non-porouscollagen-based material, and contacting a surface of the porouscollagen-based material with the gel applied to the bonding surface topartially hydrate a section of the porous material at the interfacebetween materials; b) drying the gel to bond the materials together; andc) crosslinking the collagens in the bonding layers. The fabricatedbiomaterial obtained combines a porous collagen based material, whichmay be mineralized [0042], [0048], and a mechanically strong non-porouscollagen-based material, thus providing a scaffold for regeneration ofload-bearing tissues (notably meniscus, articular cartilage, tendons andligaments), which has both porosity and mechanical strength, i.e. isable to resist compressional and tensional forces. Nothing is disclosedon the resistance to bending moments of that combined biomaterial or onthe composition of the mineralized porous collagen-based material.

US 2014/0193477 teaches that in the fabrication of collagen mats fromsoluble collagen stretching the collagen prior to its crosslinkingincreases its mechanical strength, in particular the ultimate tensilestrength, stiffness and elastic modulus (Young's modulus) (see inparticular [0109], [0110]).

Chachra et al. 1996 Effect of applied uniaxial stress on rate andmechanical effects of cross-linking in tissue-derived biomaterials inBiomaterials disclose that stretching a pericardium derived membraneprior to its crosslinking increases its tensile strength and stiffness.

The objective of the invention disclosed in EP-A1-3175869 was to providea resorbable form stable membrane for use in the oral cavity, apt toresist to pressure, shear forces and bending moments such as to supportbone formation, bone regeneration, bone repair and/or bone replacementat a non-containing bony defect site, notably in horizontal or verticalaugmentations in the maxilla or mandible, which does not have the abovedrawbacks.

EP-A1-3175869 reports that the above objective was attained by theinvention defined as a resorbable crosslinked form stable membrane (orcomposition) for use in the oral cavity which comprises a compositelayer of collagen material and inorganic ceramic particles containing1.5 to 3.5 weight parts of inorganic ceramic for 1 weight part ofcollagen material, sandwiched between two layers of elastic pretensedcollagen material, the collagen material comprising 50-100% (w/w)collagen and 0-50% (w/w) elastin.

The term “collagen material” here means a collagen-based material whichcomprises 50-100% (w/w) collagen and 0-50% (w/w) elastin. The elastincontent is here measured by desmosine/iodesmosine determinationaccording to a modification of a known method involving hydrolysis andRP-HPLC (see e.g. Guida et al. 1990 Development and validation of a highperformance chromatography method for the determination of desmosines intissues in Journal of Chromatography or Rodriguqe 2008 Quantification ofMouse Lung Elastin During Prenatal Development in The Open RespiratoryMedicine Journal). To determine the desmosine/isodesmosine content ofdry elastin, the elastin of the sponge is subjected to elastin isolationprocedures as described by Starcher et al. in 1976 (Purification andComparison of Elastin from Different Animal Species in AnalyticalBiochemistry).

That collagen material is suitably derived from tissues of naturalorigin which contain such proportions of collagen and elastin. Examplesof such tissues include vertebrate, in particular mammalian (e.g.porcine, bovine, equine, ovine, caprine, lapine) peritoneum orpericardium membrane, placenta membrane, small intestine submucosa(SIS), dermis, dura mater, ligaments, tendons, diaphragm (thoracicdiaphragm), omentum, fascie of muscles or organs. Such tissues arepreferably porcine, bovine or equine. An interesting tissue is aporcine, bovine or equine peritoneum membrane.

Usually the collagen is predominantly collagen type I, collagen type IIIor a mixture thereof. The collagen may also include a proportion ofnotably collagen type II, type IV, type VI or type VIII or anycombination of those or any collagen types.

Preferably the collagen material contains 70-90% (w/w) collagen and30-10% (w/w) elastin.

An example of a suitable starting material for preparing such a collagenmaterial is a collagen membrane from porcine, bovine or equineperitoneum or pericardium prepared by a process similar to thatdescribed in “Example” of EP-B1-1676592, or the membrane GeistlichBio-Gide® (obtainable from Geistlich Pharma A.G., Switzerland) preparedfrom porcine peritoneum by such a process.

Preferably the collagen material is derived from a porcine, bovine orequine peritoneum or pericardium membrane, small intestine mucosa (SIS)or muscle fascie.

The collagen material is generally and preferably fibrous collagenmaterial, either with a natural fibre structure or as cut collagenfibres.

However, non fibrous collagen material, such as fibrils reconstitutedfrom molecular collagen or crosslinked collagen fragments which haveenough biocompatibility and resorbability, may also be used in thecomposite layer of collagen material and inorganic ceramic particles, orin the layers of elastic pretensed collagen material provided thatcollagen material possesses sufficient mechanical stability in terms ofelastic modulus as well as maximal tensile strength (see below).

The term “resorbable” here means that the crosslinked form stablemembrane is capable of being resorbed in vivo notably through the actionof collagenases and elastases. A controlled in vivo resorbability of thecrosslinked form stable membrane is essential to healing withoutexcessive inflammation or dehiscence. The enzymatic degradation testusing collagenase from Clostridium histolicum described in detail below(Example 4, 3) gives an excellent prediction of the in vivoresorbability.

All tested prototypes of the resorbable crosslinked form stable membraneof the invention tested showed at least 10% collagen degradation (asassessed by DC Protein assay using type I collagen as standard) after 4hours, the rate of collagen degradation (lower than for the GeistlichBio-Gide® membrane) being dependent on the crosslinking conditions used.

The term “crosslinked” means that the resorbable form stable membrane orcomposition has been submitted to at least one step of crosslinking,usually chemical crosslinking (using e.g. EDC and NHS) or crosslinkingby dehydrothermal treatment (DHT), that step being performed on theassembled composite layer of collagen material and inorganic ceramicparticles sandwiched between two layers of elastic pretensed collagenmaterial usually by chemical crosslinking (using e.g. EDC and NHS) or bydehydrothermal treatment (DHT). Optionally the composite layer ofcollagen material and inorganic ceramic particles has been crosslinkedprior to its assembling into the membrane of the invention, usually bychemical crosslinking or by dehydrothermal treatment (DHT).

The term “form stable membrane for use in the oral cavity” means thatthe resorbable crosslinked membrane is capable of supporting boneformation, bone regeneration, bone repair and/or bone replacement at anon-containing dental bony defect site in a human or animal by providinga mechanical stabilization of the defect, i.e. resistance to thepressure, shear forces and bending moments that occur in the oral cavityand extraoral tissues. The form stability of the membrane of theinvention is assessed by a 3-point uniaxial bending test described indetail below (in Example 4 2): That test is similar to the methods setforth in EN ISO 178 and ASTM D6272-10, the membrane of the inventionbeing submerged in phosphate buffered saline (PBS) at a pH of 7.4 and atemperature of 37° C. That test showed that the membrane of theinvention provides a substantially stronger stabilization a PLA membraneResorb-X® (KLS Martin).

Generally, in that 3-point uniaxial bending test, the resorbablecrosslinked form stable membrane resists to a force of at least 0.20 N,preferably at least 0.30 N, for 8 mm strain.

The term “layers of elastic pretensed collagen material” means thatprior to their crosslinking the layers of collagen material have beensubmitted to a tensioning leading to an elongation or stretching of theinitial size of the layers of collagen material from the toe region intothe linear (also called elastic) region of the stress-strain curve(Roeder et al. 2002 Tensile mechanical properties of three-dimensionaltype I collagen extracellular matrices with varied microstructure in JBiomech Eng, in particular FIG. 3, page 216, or FIG. 5 of the presentapplication). Within this linear region, the elastic modulus is highestand therefore the highest stiffness can be achieved. That tensioning maybe performed radially on the collagen material pieces, e.g. by springs.The forces to be applied for such a tensioning to lead to an elongationor stretching of the collagen material into the linear region of thestress-strain curve depend on the collagen material. When the collagenmaterial is derived from porcine, bovine or equine peritoneum membrane,the tensioning leading to the linear region of the stress-stain curve ofthe collagen material may be performed radially on the collagen materialpieces, by springs tensioned between 1 and 3 N, leading to an elongationor stretching of 40 to 100% of the initial size of the layers ofcollagen material.

The term “elastic pretensed collagen material” thus means collagenmaterial that has been stretched such as to be in the linear (elastic)region of the stress-strain curve.

The elastic modulus (also called Young's modulus), i.e. the slope of thelinear region of the stress-strain curve expressed in MPa, of theelastic pretensed collagen material is generally from 1 to 1000 MPa,preferably from 2 to 150 MPa, in particular from 5 to 80 MPa.

The presence of those two layers of “elastic pretensed collagenmaterial” sandwiching the composite layer of collagen material andinorganic ceramic particles seems to be necessary for preventing thecomposite layer from breaking when the membrane is submitted to tensile,compressive, shear forces and bending moments.

Preferably one or two of the layers of elastic pretensed collagenmaterial includes holes of 5 to 500 μm. When the membrane is in placethat punctured layer of elastic pretensed collagen material will beoriented towards the bony defect, the holes allowing an easy invasion bythe bone-forming cells into the inorganic ceramic-collagen compositematerial.

The inorganic ceramic is a biocompatible material that promotes boneregeneration such as hydroxyapatite or a natural bone mineral.

A well-known natural bone mineral that promotes bone growth in dental,periodontal and maxillofacial osseous defects is Geistlich Bio-Oss®,commercially available from Geistlich Pharma AG. That hydroxyapatitebased bone mineral material is manufactured from natural bone by aprocess described in U.S. Pat. No. 5,167,961, which enables preservationof the trabecular architecture and nanocrystalline structure of thenatural bone.

Preferably the inorganic ceramic is a hydroxyapatite based natural bonemineral, such as e.g. Geistlich Bio-Oss®.

The inorganic ceramic particles have generally a size of 50 to 600 μm,preferably of 150 to 500 μm, in particular of 250 to 400 μm.

The composite of collagen material and inorganic ceramic particlescontains 1.5 to 3.5 weight parts, preferably 2.0 to 3.0 weight parts ofinorganic ceramic for 1 weight part of collagen material.

Indeed, it has been unexpectedly found that below 1.5 weight part ofinorganic ceramic for 1 weight part of collagen material or above 3.5weight parts of inorganic ceramic for 1 weight part of collagenmaterial, the membrane is not “form stable” as defined above andassessed by the 3-point uniaxial bending test described in detail below(in Example 4.2). The form stability is especially high when thecomposite of collagen material and inorganic ceramic particles contains2.0 to 3.0 weight parts of inorganic ceramic for 1 weight part ofcollagen material.

The resorbable crosslinked form stable membrane of the invention ishydrophilic, being generally completely wetted by PBS in 5 to 15minutes.

The resorbable crosslinked form stable membrane of the invention hascell adhesion properties similar to those of Geistlich Bio-Gide®, whichis well known for its good healing properties with a low rate ofdehiscence or excessive inflammation. This is indicative of good healingproperties without adverse advents such as dehiscence or excessiveinflammation.

Such good healing properties have been observed when implanting thecrosslinked form stable membrane of the invention to protect bonydefects created in the skull of rabbits.

The thickness of the resorbable crosslinked form stable membrane of theinvention is usually from 0.5 to 2.5 mm, preferably 1.0 to 2.0 mm, inparticular 1.2 to 1.8 mm.

Typical shapes and typical dimensions of the resorbable crosslinked formstable membrane of the invention are represented in FIG. 1 ofEP-A1-3175869.

The invention of EP-A1-3175869 relates to the above resorbablecrosslinked form stable for use as an implant to support in the oralcavitys bone formation, bone regeneration, bone repair and/or bonereplacement at a non-containing dental bony defect site in a human oranimal.

EP-A1-3175869 also describes a process for preparing the above definedresorbable crosslinked form stable membrane which comprises a compositelayer of collagen material and inorganic ceramic particles sandwichedbetween two layers of elastic pretensed collagen material, comprisingthe steps of:

-   (a) Preparing a composite layer of collagen material and inorganic    ceramic particles, optionally crosslinking that composite layer,-   (b) Assembling and gluing the composite layer of collagen material    and inorganic ceramic particles between two layers of collagen    material submitted to tensioning leading to a stretching of the    collagen material in the linear region of the stress-strain curve,    thereby giving a composite layer of collagen material and inorganic    ceramic particles sandwiched between two layers of elastic pretensed    collagen material, and-   (c) Crosslinking that composite layer of collagen material and    inorganic ceramic particles sandwiched between two layers of elastic    pretensed collagen material, followed by a hydrophilic making    treatment.

Step (a) may be performed by:

-   -   Producing, as inorganic ceramic particles, hydroxyapatite bone        mineral particles from cortical or cancellous bone by a process        similar to that described in U.S. Pat. No. 5,417,975 or        alternatively grinding Geistlich Bio-Oss Small Granules        (available from Geistlich Pharma AG) into smaller particles, and        submitting those particles to a sieving in the desired range        (e.g. of 150 to 500 μm or 250 to 400 μm), thereby giving sieved        hydroxyapatite bone mineral particles.    -   Preparing fibrous collagen material by        -   submitting collagen rich tissue from porcine, bovine or            equine peritoneum or pericardium to a process similar to            that described in Example of EP-B1-1676592, or alternatively            starting from the Geistlich Bio-Gide membrane (available for            Geistlich Pharma AG) obtained from porcine peritoneum by            such a process or from the intermediate product obtained            before sterilization in the industrial production of the            Geistlich Bio-Gide membrane, called here the unsterile            Geistlich Bio-Gide membrane,        -   cutting (e.g. with scissors) pieces of the thus obtained            collagen fibrous tissue, mixing those pieces of cut collagen            fibrous tissues with dry ice using a knife mill, thus giving            cut collagen fibres,        -   cutting pieces of collagen fibrous tissues with a cutting            mill with a sieve, thus giving a sieved fraction of collagen            fibre fragments.    -   Preparing a composite layer of fibrous collagen material and        hydroxyapatite bone mineral particles by        -   mixing and shaking in phosphate buffer saline PBS, 0 to 40%            by weight of the cut collagen fibres and 60 to 100% by            weight of the sieved fraction of collagen fibre fragments            obtained above,        -   adding from 1.5 to 3.5 weight parts, in particular 2.0 to            3.0 weight parts, of the sieved hydroxyapatite bone mineral            particles obtained above to 1 weight part of the fibrous            collagen obtained in the above paragraph, centrifuging at            2000 to 6000 xg, preferably 3000 to 5000 xg, pouring the            obtained pellet into a rectangular form and forming a plate            using a spatula. The composite layer of fibrous collagen            material and hydroxyapatite bone mineral particles obtained            is dried in a vacuum oven.

Crosslinking the dried composite layer of collagen material andinorganic ceramic particles at the end of (a) is not necessary but hasthe advantage that it facilitates the handling of that composite layerduring step (b).

That crosslinking may be performed using chemicals or by dehydrothermaltreatment (DHT).

Crosslinking with chemicals may be performed using any pharmaceuticallyacceptable crosslinking agent capable of giving to the crosslinked formstable membrane the required mechanical strength. Suitable suchcrosslinking agents include gluteraldehyde, glyxoal, formaldehyde,acetaldehyde, 1,4-butane diglycidyl ether (BDDGE),N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate,hexamethylene diisocyanate (HMDC), cynamide, diphenylphosphorylazide,genipin, EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and amixture of EDC and NHS (N-hydoxysuccinimide).

Crosslinking using chemicals is conveniently performed using a mixtureof EDC and NHS.

In that case, the dried composite layer of fibrous collagen material andhydroxyapatite bone mineral particles obtained above may be crosslinkedin 10-400 mM EDC and 13-520 mM NHS in a 0.1 M MES(2-(N-morpholino)-ethanesulfonic acid) and 40% ethanol solution at pH5.5 for 1 to 3 hours at room temperature. The reaction is may be thenstopped by incubating the prototypes twice in 0.1 M Na₂HPO₄ buffer at pH9.5 for 1 to 3 hours. Polar residuals may be removed by incubating theprototypes for 1 hour in a 1 M sodium chloride solution and twice for anhour in a 2 M sodium chloride solution. The chemically crosslinkedprototypes may be washed a total of 8 times for 30-60 minutes indistilled water. Drying may then be performed by carrying out byimmersion in ethanol for 15 minutes a total of 5 times, followed bythree times diethylether treatment for 5 minutes and subsequent dryingat 10 mbar and 40° C. overnight, or by lyophilisation (freezing below−10° C. and drying by conventional lyophilisation treatment).

Alternatively, cross-linking was performed by dehydrothermal treatment(DHT) at 0.1-10 mbar and 80-160° C. for 1-4 days. In this case nosubsequent drying method is necessary.

Step (b) may be performed by:

-   -   Preparing a collagen fibre glue by        -   mixing the above sieved fraction of collagen fragments in an            aqueous H₃PO₄ solution of pH 3.5 at a concentration of 3%            using a high pressure homogenizer at 1500-2000 bar, that            mixing being repeated several times,        -   neutralizing the resulting slurry to pH 7.0 by adding a            sodium hydroxide solution, concentrating by lyophilization            the collagen and homogenizing the latter by knife milling,        -   preparing the collagen fibre glue from the slurry obtained            as a 2-10% solution in phosphate buffer saline PBS of pH 7.4            by heating to 60° C. until no further particles were            visible, and    -   Using e.g. an equipment similar to that of FIG. 2, submitting        two prewetted layers of collagen material to tensioning leading        to a stretching of the collagen material in the linear region of        the stress-strain curve, thereby giving two layers of wet        elastic pretensed collagen material,    -   inserting the composite layer of collagen material and inorganic        ceramic particles obtained in (a) imbibed with the above        collagen fibre glue between the above two layers of wet elastic        pretensed collagen material,    -   using e.g. an equipment similar to that of FIG. 3, pressing        those two layers of wet elastic pretensed collagen material        against that composite layer of collagen material and inorganic        ceramic particles imbibed with the collagen fibre glue, and    -   drying the composite layer of collagen material and inorganic        ceramic particles sandwiched between two layers of wet elastic        pretensed collagen material at a temperature of 35 to 45° C.        under reduced pressure (e.g. from 20 to 1 mbar).

In the above described procedure, one or two of the prewetted layers ofcollagen material may have been subjected to a puncturing with needlessuch as to include holes of 5 to 500 μm.

In step (c), crosslinking that composite layer of collagen material andinorganic ceramic particles sandwiched between two layers of elasticpretensed collagen material, may be performed using chemicals (usinge.g. EDC and NHS) or by dehydrothermal treatment DHT.

The chemical crosslinking may be performed using any pharmaceuticallyacceptable crosslinking agent capable of giving to the crosslinked formstable membrane the required mechanical strength. Suitable suchcrosslinking agents include gluteraldehyde, glyoxal, formaldehyde,acetaldehyde, 1,4-butane diglycidyl ether (BDDGE),N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate,hexamethylene diisocyanate (HMDC), cynamide, diphenylphosphorylazide,genipin, EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and amixture of EDC and NHS (N-hydoxysuccinimide).

The crosslinking using chemicals is conveniently performed using amixture of EDC and NHS.

In that case, the composite layer of collagen material and inorganicceramic particles sandwiched between two layers of elastic pretensedcollagen material obtained above may be crosslinked in 10-400 mM EDC and13-520 mM NHS in in a 0.1 M MES (2-(N-morpholino)-ethanesulfonic acid)and 40% ethanol solution at pH 5.5 for 1 to 3 hours at room temperature.

The reaction is may be then stopped by incubating the prototypes twicein 0.1 M Na₂HPO₄ buffer at pH 9.5 for 1 to 3 hours. Polar residuals maybe removed by incubating the prototypes for 1 hour in a 1 M sodiumchloride solution and twice for an hour in a 2 M sodium chloridesolution. The chemically crosslinked prototypes may be washed a total of8 times for 30-60 minutes in distilled water. Dehydration and drying maythen be performed by immersion in ethanol for 15 min a total of 5 timesfollowed by carrying out three times diethylether treatment for 5minutes and subsequent drying at 10 mbar and 40° C. for 30 minutes, orby lyophilisation (freezing below −10° C. and drying by conventionallyophilisation treatment) without solvent treatment.

Alternatively, cross-linking was performed by dehydrothermal treatment(DHT) at 0.1-10 mbar and 80-160° C. for 1-4 days. In this case nosubsequent drying method is necessary.

The hydrophilic making treatment of step c) generally comprisesimmersing the crosslinked composite layer of collagen material andinorganic ceramic particles sandwiched between two layers of elasticpretensed collagen material hydrophilic into a physiologicallyacceptable salt solution such as a sodium chloride solution, preferablya 100-300 g/l, in particular a 150-250 g/l sodium chloride solution.

Preferably the hydrophilic making treatment comprises immersing thecrosslinked composite layer of collagen material and inorganic ceramicparticles sandwiched between two layers of elastic pretensed collagenmaterial hydrophilic into a sodium chloride solution and subsequentlydried as described by any of the methods described above.

The resorbable crosslinked form stable membrane of EP-A1-3175869 may besterilized by X-ray, beta-ray or gamma irradiation.

EP-A1-3175869 discloses various shapes of the resorbable crosslinkedform stable membrane for use in the oral cavity. That membrane may beflat (1), (1′), U-shaped straight (2), (2′) or U-shaped curved (3), (3′)corresponding to the alveolar spaces of 1 to 3 teeth (incisors, canine,premolar or molars) situated at the front, in the left-hand side orright-hand side curvature or at the rear of the denture.

It has now been found that the resorbable crosslinked form stablecomposition comprising a composite layer of collagen material andinorganic ceramic particles sandwiched between two layers of elasticpretensed collagen, which is disclosed EP-A1-3175869 can be used as aresorbable crosslinked form stable membrane for use in human tissueregeneration outside the oral cavity, notably in the craniofacial regionand the cervical, thoracic, lumbar or sacral region of the spine, or incraniotomy reconstructive surgeries, when its shape and dimensions areadapted to that use. The shapes and dimensions of the resorbablecrosslinked form stable membrane for use in the oral cavity disclosed inEP-A1-3175869 are indeed not adapted to such use outside the oralcavity.

BRIEF SUMMARY OF THE INVENTION

The invention thus concerns a resorbable crosslinked form stablemembrane which comprises a composite layer of collagen material andinorganic ceramic particles containing 1.5 to 3.5 weight parts ofinorganic ceramic for 1 weight part of collagen material, sandwichedbetween two layers of elastic pretensed collagen material, wherein theelastic pretensed collagen material is a collagen material that has beenstretched such as to be in the linear/elastic region of thestress-strain curve, the collagen material comprising 50-100% (w/w)collagen and 0-50% (w/w) elastin, and has shape and dimensions suitablefor use in human tissue regeneration outside the oral cavity inrhinoplasty, postlateral spinal fusion or orbital reconstruction.

Preferably that resorbable crosslinked form stable me is selected fromthe group consisting of:

-   -   a nasal arch-shaped membrane for rhinoplasty sized in such a way        as to fit the desired dimension of the nose,    -   an oval tube membrane for posterolateral spinal fusion with a        length such as to cover two or more vertebras and    -   a membrane for orbital fracture reconstruction shaped after        identifying the bone ledges apt to support the implant and sized        in such a way as to facilitate its insertion into the orbital        cavity.

All terms on the above definition of the invention have the samemeanings as set forth above for EP-A1-3175869.

The resorbable crosslinked form stable membrane may be a nasalarch-shaped membrane for rhinoplasty as illustrated in FIG. 1, (4), inwhich usually the length l, the width i and height h are 40 to 80 mm, 10to 15 mm and 10 to 15 mm, respectively. Generally, the thickness of thewall of the nasal arch-shaped membrane is 0.5 to 2.5 mm, preferably 1.0to 2.0 mm.

The resorbable crosslinked form stable membrane may be a slit oval tubemembrane for posterolateral spinal fusion as illustrated in FIG. 1,(5′), where usually the length l is 60 to 300 mm, an inner diameter k is5 to 10 mm and the outer diameter j of 15 to 30 mm. Generally, thethickness of the wall of oval tube membrane for posterolateral spinalfusion is 0.5 to 2.5 mm.

The resorbable crosslinked form stable membrane for use outside the oralcavity may also be a slit rectangular tube membrane for posterolateralspinal fusion as illustrated in FIG. 1, (5), which has a length l of 60to 300 mm, a width k of 5 to 10 mm and a height j of 15 to 30 mm.Generally, the thickness of the wall of slit rectangular tube membranefor posterolateral spinal fusion is 0.5 to 2.5 mm.

The resorbable crosslinked form stable membrane may be a membrane fororbital fracture reconstruction as illustrated in FIG. 1, (6) and (6′),where usually the length m, width o and height c of the membrane are 30to 50 mm, 20 to 40 mm and 5 to 25 mm, respectively. Generally, thethickness of the wall of the membrane for orbital fracturereconstruction is 0.5 to 2.5 mm.

Preferably, the composite layer of collagen material and inorganicceramic particles contains 2.0 to 3.0 weight parts of inorganic ceramicfor 1 weight part of collagen material.

Preferably, the collagen material comprises 70-90% collagen and 10-30%elastin.

The collagen material is conveniently derived from a porcine, bovine orequine peritoneum or pericardium membrane, small intestine mucosa (SIS)or muscle fascie.

Advantageously, one or both of the layers of the elastic pretensedcollagen material includes holes of 5 to 1000 μm.

Generally, the inorganic mineral particles have a size of 150 to 500 μm.

The inorganic ceramic may be selected from the group consisting ofhydroxyapatite or hydroxyapatite bone mineral.

The invention also concerns a resorbable crosslinked form stablemembrane which comprises a composite layer of collagen material andinorganic ceramic particles containing 1.5 to 3.5 weight parts ofinorganic ceramic for 1 weight part of collagen material, sandwichedbetween two layers of elastic pretensed collagen material, wherein theelastic pretensed collagen material is a collagen material that has beenstretched such as to be in the linear/elastic region of thestress-strain curve, the collagen material comprising 50-100% (w/w)collagen and 0-50% (w/w) elastin, and has shape and dimensions suitablefor use in human tissue regeneration outside the oral cavity inrhinoplasty, postlateral spinal fusion or orbital reconstruction, withthe exception of a form stable membrane for use in the oral cavity whichis flat (1), (1′), U-shaped straight (2), (2′) or U-shaped curved (3),(3′) corresponding to the alveolar spaces of 1 to 3 teeth (incisors,canine, premolar or molars) situated at the front, in the left-hand sideor right-hand side curvature or at the rear of the denture.

The invention also relates to a method of regenerating human tissueoutside of the oral cavity comprising applying the resorbablecrosslinked form stable membrane of claim 1 to a surgical site in ahuman patient undergoing rhinoplasty, postlateral spinal fusion ororbital reconstruction.

DETAILED DESCRIPTION OF THE INVENTION Rhinoplasty

Rhinoplastic corrections change the geometric dimensions of a nose fordifferent reasons such as esthetics, trauma or cancer. In case thedimension of the nose is enlarged, augmenting materials are used such asautologous cartilage, skin and bone materials or artificial materials,notably polytetrafluorethan, silicone or polyethylene implants. Forsmall augmentation collagen or hyaluronic acid fillers are generallyused (Jasin 2013 Nonsurgical rhinoplasty using dermal fillers in FacialPlast Surg Clin North Am and Malone et al. 2015 Dorsal Augmentation inRhinoplasty: A Survey and Review in Facial Plast Surg). The membrane forrhinoplasty prepared according to the invention is made for bigaugmentation, thus a direct comparison to fillers is not necessary.

The membrane for rhinoplasty prepared according to the invention issized in such a way as to fit the desired dimension of the nose. Arepresentative membrane for rhinoplasty is depicted in FIG. 1, (4). Thelength l, width i and height h of the membrane for rhinoplasty preparedaccording to the invention are generally 40-80 mm, 10-15 mm and 10-15mm, respectively. The thickness of the wall of the resorbablecrosslinked membrane for rhinoplasty is usually from 0.5 to 2.5 mm,preferably 1.0 to 2.0 mm, in particular 1.2 to 1.8 mm. The membrane forrhinoplasty prepared according to the invention may contain holesbetween 5 μm and 1000 μm to facilitate liquid uptake and bone formationinside the walls of the product. The device can be cut by the surgeon toits desired size using scissors or a scalpel. Further it can be fixedusing e.g. resorbable threads, pins or screws.

The advantages of the membrane for rhinoplasty prepared according to theinvention over the use of autologous bone, cartilage or skin include (I)its unlimited availability, (II) the reduction in surgery time, (III)the omission of donor site morbidity, (IV) the total flexibility inchoosing the shape and (V) the more predictable resorption or tissueintegration time.

The advantages of the membrane for rhinoplasty prepared according to theinvention over the use of non-natural and non-resorbable materials, suchas polytetrafluorethane, silicone or polyethylene include (I) thesuperior tissue adhesion to the membrane, thus avoiding extrudingproducts either during wound healing and over the long term use and (II)a more complication free tissue integration due to using naturalmaterials.

Posterolateral Spinal Fusion

Posterolateral spinal fusion is a common procedure to reduce back pain,especially in the lumbar region (Jacobs et al. 2013 The evidence onsurgical interventions for low back disorders, an overview of systematicreviews in Eur Spine J). Two or several vertebras are fused during asurgery by placing two pedicle screws per vertebra to be fused andconnecting the vertebras using a steel rod. Between the transversevertebras and around the steel rod, autologous bone, allogenic orxenogenic bone blocks, bone substitute putties, demineralized bonematrices, BMPs with a carrier, or sponges containing collagen andhydroxyapatite or any other suitable material are placed. The mechanicalstabilization by the steel rood and pedicle screw subsequently allow newbone formation inside the grafted area and between the vertebras (Chenget al. 2009 Posterior lumbar interbody fusion versus posterolateralfusion in spondylolisthesis: a prospective controlled study in the Hannationality in Int Orthop)

The membrane for posterolateral spinal fusion prepared according to theinvention is generally a slit rectangular or oval tube. A representativemembrane for posterolateral fusion is depicted in FIGS. 1, 5 and 5′. Thelength of the device is such that it covers 2 or more vertebras.Therefore, its length l is generally at least 6 cm. Typically the innerdiameter k of the oval device is 5 to 10 mm and the outer diameter j 15to 30 mm. Generally, for the rectangular device the width is 5-10 mm andthe height 15 to 30 mm. The thickness of the wall of the membrane forposterolateral fusion prepared according to the invention is usuallyfrom 0.5 to 2.5 mm. The membrane may contain holes between 5 μm and 1000μm to facilitate bone formation inside the product.

The device can be cut by the surgeon to its desired size using scissorsor a scalpel. Prior or after application, the device may be filled withautologous bone particles obtained during surgery and not at a secondsite, allogenic or xenogenic bone particles, bone substitute putties,demineralized bone matrices, BMPs with a carrier, or sponges containingcollagen and hydroxyapatite, BMA, blood fractions such as platelet richplasma or platelet rich fibrin or any other suitable material. Themembrane is then either fixed with screws, k-wires or resorbable threadsto the pedicle or process of the vertebra. In another variation of theoperation, the empty membrane for posterolateral fusion preparedaccording to the invention is fixed with the pedicle screw and afterfixation of the steel rod, the remaining space inside the tube is filledwith the materials described above.

The advantages of the membrane for posterolateral spinal fusion preparedaccording to the invention over the use of autologous bone blocksinclude (I) the device can be filled with the material of choice (seelist above), (II) no donor site morbidity and (III) simplification andacceleration of the surgical procedure due to the fact that only onedevice needs to be fixed per pedicle.

The advantages of the membrane for posterolateral spinal fusion preparedaccording to the invention over the use of allograft bone blocks include(I) the device can be filled with the material of choice (see listabove) and (II) simplification and acceleration of the surgicalprocedure due to the fact that only one device needs to be fixed perpedicle.

The advantages of the membrane for posterolateral fusion preparedaccording to the invention over the use of a putty material include (I)simplification and acceleration of the procedure since the device isalready shaped, (II) the use of autologous bone particles, BMA, plateletrich plasma or fibrin is enabled, (III) increased mechanical stability,thus enabling more bone formation, (IV) enabling the fixation withscrews, k-wires or resorbable threads, thus allowing for a mechanicallymore stable interface between device and vertebral bone which facilitatebone formation.

The advantages of the membrane for posterolateral fusion preparedaccording to the invention over the use of collagen/hydroxyapatitesponge, a collagen sponge with or without a growth factor or ademineralized bone matrix include (I) the device can be filled with thematerial of choice (see list above), (II) increased mechanical stabilitythus enabling more bone formation.

Orbital Fracture Reconstruction

Fracture of the orbit is most common due to vehicle accidents, assaultsand sport related injuries. Orbital fractures are generally repairedimmediately after the fracture or after two weeks, the repair procedureinvolving the use of autologous tissues or different types of implantssuch as (I) autologous bone or cartilage, (II) non-customized butadaptable materials such as titanium meshes or porous polyethylene,(III) resorbable sheetings made of e.g. polylactide acid, polyglycolicacid, polyglactin or polydioxanone, (IV) resorbable and adaptable xeno-or allo-grafts made from e.g. dura mater or demineralized human bone,(V) patient specific non-resorbable devices made from e.g. titanium,polyetheretherketone or glass-bioceramics. (Boyette et al 2015Management of orbital fractures: challenges and solutions in ClinicalOphthalmolog and Avashia et al. 2012 Materials used for reconstructionafter orbital floor fracture in J Craniofac Surg).

The access to the orbital fracture can be gained using differentmethods. The fractured site is prepared to identify the bone ledges aptto support the implant. The implant is then shaped and placed andtypically fixed using screws; implants over small defects are not fixed(Kunz 2012 Orbital fractures. Principles of Internal Fixation of theCraniomaxillofacial Skeleton in Trauma and Orthognathic Surgery byEhrenfeld et al. AO Foundation).

The membrane for orbital fracture reconstruction prepared according tothe invention is sized in such a way as to facilitate its insertion intothe orbital cavity. A representative membrane for orbital fracturereconstruction of the right orbital floor and the medial wall isdepicted in FIGS. 1, (6) and (6′). The length m, width o and height c ofthe membrane for orbital fracture reconstruction may for example be 30to 50 mm, 20 to 40 mm and 5 to 25 mm, respectively. Depending on thefracture and the dimension of the orbital cavity, membranes for orbitalfracture reconstruction of different sizes may be manufactured. A leftorbital floor and medial wall reconstruction device has the same,mirrored dimensions. Depending on the fracture, products with additionalprotuberances may be manufactured allowing fixation e.g. on the frontalaspects of the zygomatic bone.

The thickness of the wall of the resorbable crosslinked membrane fororbital fracture reconstruction prepared according to the invention isusually from 0.5 to 2.5 mm. The membrane for orbital fracturereconstruction may contain holes between 5 μm and 1000 μm to facilitateliquid uptake and bone formation inside the walls of the product. Thedevice may be cut by the surgeon to its desired size using scissors or ascalpel. Further it can be fixed using e.g. resorbable threads, pins orscrews. The advantages of the membrane for orbital fracturereconstruction prepared according to the invention over autologous boneor cartilage include (I) its unlimited availability, (II) reduction insurgery time, (III) the omission of donor site morbidity, (IV) apredictable time until it is fully ossified and at least for bone abetter adaptability to the contours of the orbital fracture and (V) itsradio-opacity compared to cartilage, thus enabling the control ofimplant position by computed tomography. The advantages of the membranefor orbital fracture reconstruction prepared according to the inventionover non-customized but adaptable materials such as titanium meshes orporous polyethylene include (I) its design to become ossified andresorbed or osseo-integrated over time (II) smooth edges which do notjeopardize the surrounding soft tissues (III) in comparison tonon-radiopaque materials, e.g. porous poly ethylene, its radio-opacity,thus enabling the control of implant position by computed tomography.

The advantages of the membrane for orbital fracture reconstructionprepared according to the invention over resorbable sheets made of e.g.polylactide acid, polyglycolic acid, polyglactin or polydioxanoneinclude (I) its design to become ossified and resorbed orosseo-integrated over time (osteoconductive) (II) the fact that noacidic degradation products apt to cause inflammation and local boneresorption are released during resorption and (III) its radio-opacity,thus enabling the control of implant position by computed tomography.

The advantages of the membrane for orbital fracture reconstructionprepared according to the invention over resorbable and adaptable xeno-or allo-grafts made from e.g. dura mater or demineralized human boneinclude (I) its higher form stability upon bending allowing its use totreat larger fractures and (II) its radio-opacity, thus enabling thecontrol of implant position by computed tomography.

Interesting features of the resorbable crosslinked form stable membranefor use in human tissue regeneration outside the oral cavity includethose disclosed in EP-A1-3175869 in particular that

-   -   the composite layer of collagen material and inorganic ceramic        particles contains 2.0 to 3.0 weight parts of inorganic ceramic        for 1 weight part of collagen material,    -   the collagen material comprises 70-90% (w/w) collagen and 10-30%        (w/w) elastin,    -   the collagen material is derived from tissues of natural origin        selected from the group of mammalian peritoneum or pericardium        membrane, placenta membrane, small intestine submucosa (SIS),        dermis, dura mater, ligaments, tendons, diaphragm (e.g. thoracic        diaphragm), omentum and fascie of muscles or organs,    -   the collagen material is derived from a porcine, bovine or        equine peritoneum or pericardium membrane, small intestine        mucosa (SIS) or muscle fascie,    -   the elastic pretensed collagen material has an elastic modulus        of 2 to 150 MPa, in particular from 5 to 80 MPa,    -   one or both of the layers of the elastic pretensed collagen        material includes holes of 5 to 1000 μm,    -   the inorganic mineral particles have a size of 150 to 500 μm,    -   the inorganic ceramic is hydroxyapatite,    -   the inorganic ceramic is hydroxyapatite bone mineral,    -   the form stable membrane is chemically crosslinked, and/or    -   the form stable membrane is crosslinked by dehydrothermal        treatment DHT. The form stable membrane may be used in        combination with growth factors and/or cartilage- or        bone-forming cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in further detail hereinafter withreference to illustrative examples of preferred embodiments of theinvention and the accompanying drawing figures, in which:

FIG. 1 represents typical shapes and typical dimensions of resorbablecrosslinked form stable membranes:

-   -   For use in the oral cavity according to the invention of        EP-A1-3175869. Those membranes may be flat (1), (1′), U-shaped        straight (2), (2′) or U-shaped curved (3), (3′) corresponding to        the alveolar spaces of 1 to 3 teeth (incisors, canine, premolar        or molars) situated at the front, in the left-hand side or        right-hand side curvature or at the rear of the denture. The        size of the anterior products is similar to that of the        posterior products, the radius of the curvature being such as to        conform to the alveolar ridge. Typical dimensions are a=5-20 mm,        b=8-20 mm, c=6-10 mm, d=25-40 mm, e=15 mm, f=20-40 mm.    -   For use in human tissue regeneration outside the oral cavity        according to the present invention:    -   the arch-shaped membrane for rhinoplasty (4),    -   the slit rectangular or oval tube membrane for posterolateral        spinal fusion (5), (5′), respectively, and    -   a membrane for orbital fracture reconstruction (6) and (6′)        (front view and plan view, respectively.

Typical dimensions of membrane for rhinoplasty (4) are: 1=40-80 mm,i=10-15 mm and h=10-15 mm. The rostral ridge of the device may come indifferent shapes.

Typical dimensions of a membrane for posterolateral spinal fusion (5 and5′) are: 1=60-300 mm, j=15-30 mm and k=5-10 mm. The shape of theposterolateral spinal fusion device may be rectangular (5) or oval (5′).

(6) and (6′) show a front view and a plan view of a representativemembrane for orbital fracture reconstruction. Typical dimensions are:m=30-50 mm, o=20-40 mm and c=5-25 mm.

FIG. 2 is a schematic view of equipment suitable for enabling thetensioning of the polymer layers prior to their assembling into a flator U-shaped form stable membrane prepared according to the invention.

FIG. 3 represents the assembly of a flat form stable membrane, wherein(1) is a steel plate, (2) is a compressed polyurethane sponge, (3) is apolyamide net, (4) is a layer of elastic pretensed collagen and (5) is acrosslinked hydroxyapatite-collagen plate.

FIG. 4 represents the variation of the force as a function of the strainin a 3-point bending analysis test for the resorbable form stablemembrane of the invention crosslinked by EDC/NHS or DHT in comparison tothe PLA membrane Resorb-X® (KLS Martin).

FIG. 5 represents the stress-strain curves of a few commerciallyavailable, wet and sterile collagen materials that could be used in thelayers of elastic pretensed collagen material of the resorbablecrosslinked form stable membranes according to the invention, namelyporcine peritoneum derived Geistlich Bio-Gide® collagen membrane(Geistlich Pharma AG), porcine pericardium derived Jason® collagenmembrane (aap Biomaterials/Botiss) and porcine SIS derived Dynamatrix®collagen membrane (Cook Biotech Inc.), and a collagen material derivedfrom muscle fascie. In each of those stress-curves there is a toe regioncharacterized by large strains upon minimal values of stress, a linearor elastic region characterized by a linear increase in strain per unitstress and a failure region characterized by rupture of polymericfibres. In the stress-stain curves represented in this figure, theelastic modulus (or Young's modulus, i.e. the slope of the linear regionof the stress-strain curve) is about 8 MPa for the Geistlich Bio-Gide®membrane, about 64 MPa for the Jason membrane, about 54 MPa for theDynamatrix® membrane and about 56 MPa for the collagen material derivedfrom muscle fascie.

FIG. 6 is a column diagram of the % of human gingival fibroblasts thathave adhered to the membrane after incubation for 24 hours at 37° C. forGeistlich Bio-Gide® collagen membrane, a prototype of the resorbableform stable membrane of the invention crosslinked by DHT (FRM) and theCystoplast® PTFE membrane (Keystone Dental).

The following examples illustrate the invention without limiting itsscope.

EXAMPLE 1 PREPARATION OF THE RAW MATERIALS

Preparation of Hydroxyapatite Fine Particles Having a Size of 250 to 400μm (A)

Hydroxyapatite bone mineral fine particles were produced from corticalor cancellous bone as described in Examples 1 to 4 of U.S. Pat. No.5,417,975, using an additional sieving step between 250 and 400 μm.

Alternatively, hydroxyapatite bone mineral fine particles were producedby grinding Geistlich Bio-Oss® Small Granules (available from GeistlichPharma AG, CH-6110, Switzerland) by careful impactation using a pistoland an additional sieving step between 250 and 400 μm.

The hydroxyapatite bone mineral fine particles having a size of 250 to400 μm prepared above (A) were stored in glass bottles until use.

Preparation of Collagen Fibres (B)

As described in “Example” of EP-B1-1676592, peritoneal membranes fromyoung pigs were completely freed from flesh and grease by mechanicalmeans, washed under running water and treated with 2% NaOH solution for12 hours. The membranes were then washed under running water andacidified with 0.5% HCl. After the material had been acidified throughits entire thickness (for about 15 minutes) the material was washed withwater until a pH of 3.5 was obtained. The material was then shrunk with7% saline solution, neutralised with 1% NaHCO₃ solution and washed underrunning water. The material was then dehydrated with acetone anddegreased with n-hexane and dried using ethanol ether. 2×2 cm pieces ofthe collagen membranes thus obtained were cut by hand using scissors.

Alternatively, 2×2 cm pieces of the Geistlich Bio-Gide® membrane(available from Geistlich Pharma AG) were cut by hand using scissors.

1 g of the 2×2 cm pieces of the collagen membranes obtained above wasmixed with 200 ml of dry ice and mixed in a knife mill (Retsch®Grindomix) at 5000 rpm until no blockage occurred. The speed was thenincreased to 6000, 7000, 9000 and 10′000 rpm for 20 to 30 seconds, eachtime adding 50 ml of dry ice.

The dry ice was evaporated and the collagen fibres thus obtained (B)were stored in Minigrip plastic wraps until further use.

Preparation of Cutting Mill Collagen Fibre Segments (C)

The 2×2 cm collagen fibre pieces obtained above were cut in a cuttingmill with a 0.8 mm sieve at 1500 rpm, giving a sieved fraction ofcutting mill collagen fibre segments (C).

Preparation of a Collagen Fibre Glue (D)

The sieved fraction of cutting mill collagen fibre segments (C) wasmixed in water to obtain a solution of 3%, the pH was set to 3.5 byadding phosphoric acid H₃PO₄ and the suspension was high pressurehomogenized at 1500-2000 bar, this being repeated 3 to 5 times.

The resulting slurry was neutralized to about pH 7 by adding a sodiumhydroxide solution NaOH and gelled overnight at 4° C. The collagen wasconcentrated by lyophilisation at −10° C. and 0.310 mbar after freezingfor 4 hours at −40° C. and homogenized by knife milling.

The collagen fibre glue (D) was prepared from the slurry obtained as a2-10% solution in phosphate buffered saline, pH 7.4 by heating to 60° C.until no further particles were visible.

EXAMPLE 2 PREPARATION OF AN OPTIONALLY CROSSLINKEDHYDROXYAPATITE/COLLAGEN PLATE (E)

4 g of collagen fibres (B) and 6 g of cutting mill collagen fibresegments (C) prepared in Example 1 were mixed with 140 g of phosphatebuffered saline and shaked in a cocktail mixer. In another example,collagen fibres were substituted completely by cutting mill collagenfibre segments.

20 to 30 g hydroxyapatite fine particles (A) prepared in Example 1 wereadded and mixed by hand.

34.14 g of this mixture were centrifuged at 7000×g (7000 times theacceleration of gravity) for 2 minutes.

The pellet was poured between two polyamide-nets (of pore size 21 μm anda total of 17% of open structure) in a flat rectangular form of 8×12 cmand the matter was condensed by removing excess water with a laboratoryspoon. The plates obtained were compressed at a pressure of 1-1.7 kPaand dried in a vacuum oven at 30° C./50 mbar for 2 hours, then at 30°C./10 mbar for 8 hours. The polyamide-nets were removed.

Optional Crosslinking of the Hydroxyapatite-Collagen Plate

To facilitate handling of the hydroxyapatite-collagen plate, the latterwas crosslinked chemically or by dehydrothermal treatment (DHT).

Chemical cross-linking of the collagen with EDC/NHS was performed,leading to an increase of overall stability of thehydroxyapatite-collagen plate plates. The dried plates were thencross-linked in 10-400 mM EDC and 13-520 mM NHS in 0.1 M MES(2-(N-morpholino)-ethanesulfonic acid) and 40% ethanol at pH 5.5 for 2hours at room temperature.

The reaction was stopped by incubating the prototypes twice in 0.1 MNa₂HPO₄ buffer at pH 9.5 for an hour. Polar residuals were removed byincubating the prototypes for 1 hour in a 1 M sodium chloride solutionand twice for an hour in a 2 mol/1 sodium chloride solution. Thechemically crosslinked prototypes were washed a total of 8 times for30-60 minutes in distilled water, then dehydrated by immersion inethanol for 15 minutes a total of 5 times. Drying was then performed bycarrying out three times diethylether treatment for 5 minutes andsubsequent drying at 10 mbar and 40° C. for 30 minutes, or bylyophilisation (freezing below −10° C. and drying by conventionallyophilisation treatment).

Alternatively, cross-linking was performed by dehydrothermal treatment(DHT) at 0.1-10 mbar and 80-120° C. for 1-4 days. In this case nosubsequent drying method was necessary.

EXAMPLE 3 PREPARATION OF A RESORBABLE CROSSLINKED FORM STABLE MEMBRANE(M) BY ASSEMBLING AND GLUING TWO ELASTIC PRETENSED COLLAGEN LAYERS ONTHE TWO OPPOSITE FACES OF THE HYDROXYAPATITE/COLLAGEN PLATES (E)

The following description will be better understood by referring toFIGS. 2 and 3. The assembly of a flat or U-shaped prototype requires theuse of fixed or bendable frames enabling the tensioning of the layers ofcollagen material.

Forming of Flat or U-Shaped Prototypes (F)

FIG. 2 is a schematic view of equipment suitable for enabling thetensioning of the layers of collagen material prior to their assemblinginto a flat or U-formed form stable membrane of the invention.

That equipment consists of a frame (a), which can be made of anysuitable material, e.g. steel or aluminum. The main purpose for theframe is to anchor the springs (b), which tension the two wet collagenlayers (c). The hydroxyapatite/collagen plate (E) was positioned inbetween the two collagen layers (c).

If a U-shaped resorbable crosslinked form stable membrane is desired, anegative form (e) for bending the collagen plate (E) and frames withhinges (f) are used, thus leading to U-shaped straight prototypes.

Collagen material layers of unsterile Geistlich Bio-Gide Collagen layerswere pretensed by elongating or stretching 40 to 100% of initial lengththrough tensioning each spring by 2-3 N, such as to be in the linearregion of the stress-curve of the collagen material. Within this linearregion, the elastic modulus is highest and therefore the higheststiffness is achieved

Due to the viscoelastic nature of collagenous tissues, wet and tensionedmaterials were kept for approximately 30 minutes in tensioned state. Dueto the relaxing of the pretensed collagen membrane, the springs weretensioned again to 1-3 N, such as to be in the linear region of thestress-curve of the collagen material.

Two round pieces of collagen with a diameter of 10 cm cut from unsterileGeistlich Bio-Gide® collagen membrane were used, one of which waspunctured with a needle drum containing 50 needles per cm² with a shaftdiameter of 0.88 mm. Those two round pieces of collagen were wetted andtensioned in a radial manner by 12 springs each tensioned to 1-3 N,leading to an elongation of 40-100% of the initial size of the collagenpieces.

Upon completion of this step, the hydroxyapatite/collagen plates (E)were wetted on both faces with the collagen fibre glue (C) and then, thehydroxyapatite/collagen plate was placed between the two elasticpretensed collagen layers. The central bar (e) as well as the hinges (f)are necessary to produce U-shaped prototypes (see below).

The elastic pretensed membranes were placed on a heating plate andprewarmed to 40° C.

The cross-linked Bio-Oss plate (E) obtained in Example 2 was shortlysubmerged in prewarmed collagen fibre glue (D) and placed between thetwo elastic pretensed collagen membranes.

Polyamide nets, as well as sponges (of thickness 5 cm, density ofapprox. 20-25 mg/cm³, containing interconnected pores, made ofpolyurethane), were placed on both sides, compressed by 50-95% leadingto compression pressures of up to 120 kPa.

See FIG. 3, which represents the assembly of a flat form stablemembrane, wherein (1) is a steel plate, (2) is a compressed polyurethanesponge, (3) is a polyamide net, (4) is a layer of elastic pretensedcollagen and (5) is a crosslinked hydroxyapatite-collagen plate.

Subsequently, the construct was dried in a vacuum oven at 40° C. with asteady decrease in air pressure down to 10 mbar for a total of 32 hours.

Forming of U-Shaped Prototypes

The skilled person will readily adapt the apparatus of FIGS. 2 and 3 andthe method described above to the forming of U-shaped prototypesstraight or curved, by bending the construct over an appropriatenegative form and replacing one of the sponges by a thinner polyurethanesponge or a fibre free paper towel.

Cross-Linking of Flat or U-Shaped Prototypes (G)

Flat or U-shaped prototypes (F) were cut into the desired dimensionsusing scissors or a small circular saw. The prototypes were thencrosslinked chemically or by dehydrothermal treatment (DHT).

Chemical crosslinking was performed in 0.1 M MES buffer at pH 5.5 and anethanol content of 40 Vol-% at concentration of EDC and NHS of 10 to 400mM and 13 to 520 mM respectively. The prototype concentration in thecross-linking solution was 10%. To enable homogenous cross-linking,plates were initially treated under vacuum (<40 mbar) and thecross-linking reaction was carried out at 4° C. for 2 hours, all buffersbeing precooled to this temperature.

The reaction was stopped by incubating the prototypes twice in 0.1 MNa₂HPO₄ buffer at pH 9.5 for an hour. Polar residuals were removed byincubating the prototypes for 1 hour in a 1 M NaCl solution and twicefor an hour in a 2 M NaCl solution. Prototypes were washed a total of 8times for 30-60 minutes in distilled water. Dehydration and drying wasthen performed by carrying out 5 times ethanol treatment for 15 minutesand three times diethylether treatment for 5 minutes and subsequentdrying at 10 mbar and 40° C. overnight or until the product wascompletely dry, or by conventional lyophilisation (freezing below −10°C. and drying by conventional lyophilisation treatment) of the not bysolvent treated product. Alternatively, cross-linking was performed bydehydrothermal treatment (DHT) at 0.1-10 mbar at 80-160° C. for 1-4days. In this case no subsequent drying method was necessary.

Prototypes obtained by the above described methods are wetted in salineor PBS within an hour or two. To allow wetting within 10 min, prototypesare rewetted in distilled water for approximately 1 to 2 hours. At thistime the perforation of one or two sides with the above described needledrum is possible too. Sodium chloride is applied by incubating theprototypes three times for 40 min in a 200 g/1 NaCl solution. The sodiumchloride is precipitated as described below (H).

Drying of Cross-Linked Flat or U-Shaped Prototypes (H)

The crosslinked prototypes were dehydrated by immersion in ethanol for15 minutes a total of 5 times. They were then dried by either solventdrying (three times diethylether treatment for 5 minutes and subsequentdrying at 10 mbar and 40° C.) or conventional lyophilisation (freezingbelow −10° C. and drying by conventional lyophilisation treatment).

The thickness of the crosslinked form stable membrane of the differentprototypes in wet state was from 1.0 to 2.0 mm, for most of them from1.2 to 1.8 mm.

The dried prototypes were optionally sterilized by x-ray-irradiation at27-33 kGy.

EXAMPLE 4 PROPERTIES OF THE RESORBABLE CROSSLINKED FORM STABLE MEMBRANE

The following characteristics of the resorbable cross-linked form stablemembrane obtained in Example 3 were determined: (1) Wettability in PBS,(2) Mechanical strength, (3) Enzymatic degradation using collagenasefrom Clostridium histolyticum and (4) Cell adhesion (5) Measurement ofthe elongation of the elastic pretensed collagen material layers (6)Measurement of the thickness of the collagen-hydroxyapatite plates andfinal prototypes

(1) Wettability in PBS

The time of complete wetting in PBS (Phosphate buffer saline) asassessed visually was observed to be between 5 and 10 minutes for thedifferent prototypes of the resorbable crosslinked form stable membrane,that time depending mainly on the treatment with sodium chloride priorto dehydration with ethanol and drying.

(2) Mechanical Strength

The form stability of the membrane of the invention was assessed by a3-point uniaxial bending test which is similar to the methods describedin EN ISO 178 and ASTM D6272-10, the membrane of the invention beingsubmerged in PBS at a pH of 7.4 and a temperature of 37° C.

This test was considered most useful, because every form stable membranedesigned to mechanically stabilize a bony defect at a non-containingsite will experience bending moments. Therefore, 3- or 4-point bendingcan be used as a test to characterize the used materials andadditionally to compare different products with e.g. differentthicknesses. For material characterization, the bending modulus is themost suitable parameter. However, to compare different products whichhave different thicknesses, the maximal force after 8-10 mm ofindentation is more relevant and therefore used, to characterize theproduct.

In the 3-point uniaxial bending test used, the specimens were cut to asize of 50×13 mm and incubated in PBS at 37° C. until complete wettingas visually observed. Mechanical testing was conducted at 5 mm perminutes in a 3-point bending apparatus with a support span width of 26mm and a radius of 5 mm of each supporting structure. The bending modulewas calculated within 1 and 5% bending strain. The resulting maximalforces were read out after lowering the central indenter between 8 and10 mm.

The test was performed for a membrane of the invention of thickness 1.5mm crosslinked by EDC/NHS, a membrane of the invention of thickness 1.6mm crosslinked by DHT and the PLA membrane Resorb-X® from KLS Martin ofthickness 0.137 mm.

FIG. 4, which represents the variation of the force as a function of thestrain for those membranes, shows that the mechanical stability ofmembrane of the invention crosslinked by EDC/NHS (about 0.65 N for 8 mmstrain) or crosslinked by DHT (about 0.40 N for 8 mm strain) issubstantially superior to that of the PLA membrane Resorb-X® (about 0.10N for 8 mm strain). The membrane of the invention will thus betterstabilize the bony defect at a non-containing site.

(3) Enzymatic Degradation Test Using Collagenase from Clostridiumhistolyticum

In the human body collagens are degraded by human tissuematrix-metalloproteinase (MMP), cathepsins and putatively by some serineproteinases. Best studied are the MMPs where collagenases (notablyMMP-1, MMP-8, MMP-13 and MMP-18) are the most important enzymes fordirect collagen degradation (Lauer-Fields et al. 2002 Matrixmetalloproteinases and collagen catabolism in Biopolymers—PeptideScience Section and Song et al. 2006 Matrix metalloproteinase dependentand independent collagen degradation in Frontiers in Bioscience).

Collagenase capability to degrade collagen tissues and membranes dependson the substrate flexibility and collagen type, MMP active sites and MMPexosites. Collagenases align at the triple helical collagen, unwind itand subsequently cleave it (Song et al. 2006, see above).

With the view of overcoming differences in degradation between thedifferent collagen types, collagenase degradation of collagen is oftenassessed using collagenase from Clostridium histolyticum which has ahigh catalytic speed (Kadler et al. 2007 Collagen at a glance in J CellSci). Generally, a natural collagen product degrades faster than achemically cross-linked collagen product.

In this test the collagen products (samples of the resorbablecross-linked form stable membrane at 1 mg/ml collagen) were incubated at37° C. with 50 units/ml from Clostridium histolyticum (one unit beingdefined as liberating peptides from collagen from bovine Achilles tendonequivalent in ninhydrin color to 1.0 micromole of leucine in 5 hours atpH 7.4 at 37° C. in the presence of calcium ions) in a calciumcontaining Tries-buffer and the degradation of the collagen matrix wasmeasured visually and with the “DC Protein Assay” from Bio-RadLaboratories (Hercules, USA, Order Nor. 500-0116) using Collagen Type Ias reference material. The collagen concentration was determined using amicrowellplate spectrometer (Infinite M200, available from Tecan).

All prototypes of the resorbable crosslinked form stable membrane of theinvention showed at least 10% collagen degradation (as assessed by DCProtein assay using collagen type I as standard.) after 4 hours, therate of collagen degradation (lower than for the Geistlich Bio-Gide®membrane) being dependent on the crosslinking conditions used.

(4) Cell Adhesion

Cell adhesion to different membranes was assessed by first seeding 8 mmmembrane punches with 100′000 human gingival fibroblasts previouslylabelled with a fluorescent, lipophilic dye, incubating for 24 hours at37° C., removing non-adherent cells by washing the membranes in PBS,lysing adherent cells and quantifying them by measuring fluorescence at485 nm. Fluorescence was normalized to a standard curve established withcell-seeded membrane punches that were not washed prior to lysis.

The results obtained for the form stable resorbable membrane arerepresented in FIG. 5 which is a column diagram representinghorizontally the % of cells capable to adhere on different types ofdental membranes in percentage, the resorbable crosslinked form stablemembrane of the invention and the Cystoplast® PTFE membrane (KeystoneDental).

FIG. 5 shows that adhesion to the resorbable crosslinked form stablemembrane of the invention is about 10.5%, a value much closer to that ofthe Geistlich Bio-Gide® membrane of about 13% than to that of theCystoplast® PTFE membrane of about 4%. The Geistlich Bio-Gide® membraneis well known for its good healing properties with a low rate ofdehiscence (Zitzmann et al. 1997 Resorbable versus non-resorbablemembranes in combination with Bio-Oss for guided bone regeneration inInt J Oral Maxillofac Implants and Tal et al. 2008 Long-termbio-degradation of cross-linked and non-cross-linked collagen barriersin human guided bone regeneration in Clin Oral Implants Res) or noexcessive inflammation (Jung et al. 2013 Long-term outcome of implantsplaced with guided bone regeneration (GBR) using resorbable andnon-resorbable membranes after 12-14 years in Clin Oral Implants Res)This measured value of adhesion of human gingival fibroblasts to theresorbable crosslinked form stable membrane of the invention ispredictive for soft tissue healing without adverse advents such asexcessive inflammation or dehiscence.

(5) Measurement of the Elongation of the Elastic Pretensed CollagenMaterial Layers

To determine the amount of tensioning of the collagen layers, the drycollagen layer is mounted to a tensioning ring (FIG. 2, part a) usingthe not yet tensioned springs (FIG. 2, part b). In the centre of themembrane at least 4 points, which are several centimetres apart fromeach other, are marked using a pencil or pen. The distance between eachpoint is measured using a ruler. The measured distances are defined asthe initial lengths between each point. The collagen layer is submergedin water and tensioned to the desired force. The collagen layer isincubated in water for 30 minutes. Due to the viscoelastic nature ofmost collagen layers, the tension reduces. Therefore, the collagenlayers need to be tensioned again. After 30-40 minutes of incubation thedistance between each point is measured with a ruler. The percentage ofstrain is determined by subtracting the initial length from the lengthafter tensioning, divided by the initial length multiplied by 100.

Typical results such as to be in the linear region of the stress-straincurve are between 40 and 100% strain (elongation, stretching) forunsterile Geistlich Bio-Gide. Strain values measured by this method arenot directly comparable to strain values obtained in a uniaxialelongation test.

(6) Measurement of the Thickness of Collagen Hydroxyapatite Plate andFinal Prototype

The thickness of the final prototypes or the collagen/hydroxyapatiteplate “E” can be measured as described above or by using a slidingcalliper.

(7) Analysis of the Mechanical Properties of Different Collagen Layers(FIG. 5)

To compare different sources of collagen layers and estimate theirmechanical properties, standard uniaxial tensioning of wet samples wasused. A general setup for such an analytical method is described in ASTMD882-09 “Standard Test Method for Tensile Properties of Thin PlasticSheeting”. Due to the high costs of the collagen membranes used, severalparameters of the testing were adapted. Samples were cut intorectangular sheets of e.g. 2×1 cm, prewetted in isotonic phosphatebuffered saline and mounted to a tensile testing machine with a distanceof 1 cm between each sample holder. The samples were tensioned at aconstant speed of 33% of initial length per minute. The prepressure, atwhich 100% initial length is recorded, was typically set to 50 kPa. Theelongation of the sample was calculated using the distance between thetwo sample holders.

The stress-strain curves of FIG. 5 were thus obtained.

EXAMPLE 5 PREPARATION OF PROTOTYPES OF THE RESORBABLE CROSSLINKED FORMSTABLE MEMBRANE FOR USE OUTSIDE THE HUMAN CAVITY

A resorbable crosslinked form stable nasal arch-shaped membrane wasprepared as described below by assembling and gluing two layers ofelastic pretensed collagen material on the two opposite faces of thehydroxyapatite/collagen plates (E)

The following description will be better understood by referring toFIGS. 2 and 3.

The assembly of a nasal arch-shaped membrane requires the use ofbendable frames enabling the tensioning of the layers of collagenmaterial.

Forming of Nasal Arch-Shaped Membrane (Membrane for Rhinoplasty),Orbital Fracture Membrane or Posterolateral Spinal Fusion MembranePrototypes

FIG. 2 is a schematic view of equipment suitable for enabling thetensioning of the layers of collagen material prior to their assemblinginto a nasal arch-shaped membrane prototype of the invention.

That equipment consists of a frame (a), which can be made of anysuitable material, e.g. steel or aluminum. The main purpose for theframe is to anchor the springs (b), which tension the two wet collagenlayers (c). The hydroxyapatite/collagen plate (E) was positioned inbetween the two collagen layers (c).

For a nasal arch-shaped membrane prototype, a negative form (e) forbending the collagen plate (E) and frames with hinges (f) are used, thusleading to nasal arch-shaped membrane.

Collagen material layers of unsterile Geistlich Bio-Gide Collagen layerswere pretensed by elongating or stretching 40 to 100% of initial lengththrough tensioning each spring by 2-3 N, such as to be in the linearregion of the stress-curve of the collagen material. Within this linearregion, the elastic modulus is highest and therefore the higheststiffness is achieved

Due to the viscoelastic nature of collagenous tissues, wet and tensionedmaterials were kept for approximately 30 minutes in tensioned state. Dueto the relaxing of the pretensed collagen membrane, the springs weretensioned again to 1-3 N, such as to be in the linear region of thestress-curve of the collagen material.

Upon completion of this step, the hydroxyapatite/collagen plates (E)were wetted on both faces with the collagen fibre glue (C) and then, thehydroxyapatite/collagen plate was placed between the two elasticpretensed collagen layers. The central bar (e) as well as the hinges (f)are necessary to produce nasal arch-shaped membrane prototypes (seebelow).

The Bio-Oss plate (E) obtained in Example 2 was shortly submerged inprewarmed collagen fibre glue (D) and placed between the two elasticpretensed collagen membranes.

Polyamide nets, as well as sponges (of thickness 5 cm, density ofapprox. 20-25 mg/cm³, containing interconnected pores, made ofpolyurethane), were placed on both sides, compressed by 50-95% leadingto compression pressures of up to 120 kPa.

See FIG. 3, which represents the assembly of a flat form stablemembrane, wherein (1) is a steel plate, (2) is a compressed polyurethanesponge, (3) is a polyamide net, (4) is a layer of elastic pretensedcollagen and (5) is a crosslinked hydroxyapatite-collagen plate.

Subsequently, the construct was dried in a vacuum oven at 40° C. with asteady decrease in air pressure down to 10 mbar for a total of 32 hours.

The skilled person will readily adapt the apparatus of FIGS. 2 and 3 andthe method described above to the forming of nasal arch-shaped membraneprototypes, the orbital fracture membrane prototype or theposterolateral spinal fusion membrane prototype, by bending theconstruct over an appropriate negative form and replacing one of thesponges by a thinner polyurethane sponge or a fibre free paper towel.

Cross-Linking of the Nasal Arch-Shaped Membrane, Orbital FractureMembrane or Posterolateral Spinal Fusion Membrane Prototypes

Nasal arch-shaped membrane prototypes, the orbital fracture membraneprototype or the posterolateral spinal fusion membrane prototype (F)were cut into the desired dimensions using scissors or a small circularsaw. The prototypes were then crosslinked chemically or bydehydrothermal treatment (DHT).

Cross-linking was performed by dehydrothermal treatment (DHT) at 0.1-10mbar at 80-160° C. for 1-4 days. In this case no subsequent dryingmethod was necessary. Prototypes obtained by the above described methodsare wetted in water, saline or PBS within an hour or two. Prototypes arecut to its final shape and are punctured with a needle bed (as describedabove). At this time the perforation of one or two sides with anappropriate needle bed is possible too.

Drying of Crosslinked Nasal Arch-Shaped Membrane, Orbital FractureMembrane or Posterolateral Spinal Fusion Membrane Prototypes

The crosslinked prototypes were dehydrated by immersion in ethanol for15 minutes a total of 5 times. They were then dried at 10 mbar and 40°C. in a vacuum oven.

Another option is to freeze dry the with water wet device (freezingbelow −10° C. and drying by conventional lyophilisation treatment).

The thickness of the crosslinked form stable membrane of the differentprototypes in wet state was from 1.0 to 2.0 mm, for most of them from1.2 to 1.8 mm.

The dried prototypes were optionally sterilized by x-ray-irradiation at27-33 kGy.

While the invention has been illustrated and described in details in thedrawings and forgoing description, such illustration and description areto be considered illustrative or exemplary and not restrictive: theinvention is not limited by the disclosed embodiments.

Other variations to the disclosed embodiments can be understood andeffected by those skilled in the art in practicing the claimedinvention, from a study of the drawings, the disclosure and the amendedclaims.

In the claims, the word “comprising” does not exclude other elements;the definite article “a” or “an” does not exclude a plurality.

1. A resorbable crosslinked form stable membrane which comprises acomposite layer of collagen material and inorganic ceramic particlescontaining 1.5 to 3.5 weight parts of inorganic ceramic for 1 weightpart of collagen material, sandwiched between two layers of elasticpretensed collagen material, wherein the elastic pretensed collagenmaterial is a collagen material that has been stretched such as to be inthe linear/elastic region of the stress-strain curve, the collagenmaterial comprising 50-100% (w/w) collagen and 0-50% (w/w) elastin, andhas shape and dimensions suitable for use in human tissue regenerationoutside the oral cavity in rhinoplasty, postlateral spinal fusion ororbital reconstruction.
 2. A resorbable crosslinked form stable membraneaccording to claim 1, which is selected from the group consisting of: anasal arch-shaped membrane for rhinoplasty sized in such a way as to fitthe desired dimension of the nose, an oval tube membrane forposterolateral spinal fusion with a length such as to cover two or morevertebras and a membrane for orbital fracture reconstruction shapedafter identifying the bone ledges apt to support the implant and sizedin such a way as to facilitate its insertion into the orbital cavity. 3.A resorbable crosslinked form stable membrane according to claim 2,which is a nasal arch-shaped membrane for rhinoplasty as illustrated inFIG. 1, (4), the length 1, the width i and height h of the membrane forrhinoplasty being 40 to 80 mm, 10 to 15 mm and 10 to 15 mm,respectively.
 4. A resorbable crosslinked form stable membrane accordingto claim 3, wherein the thickness of the nasal arch-shaped membrane is0.5 to 2.5 mm.
 5. A resorbable crosslinked form stable membraneaccording to claim 3, wherein the thickness of the wall of the nasalarch-shaped membrane is 1.0 to 2.0 mm.
 6. A resorbable crosslinked formstable membrane according to claim 2, which is a slit oval tube membranefor posterolateral spinal fusion as illustrated in FIG. 1, (5′) with alength l of 60 to 300 mm, an inner diameter k of 5 to 10 mm and an outerdiameter j of 15 to 30 mm.
 7. A resorbable crosslinked form stablemembrane according to claim 6 wherein the thickness of the membrane is0.5 to 2.5 mm.
 8. A resorbable crosslinked form stable membraneaccording to claim 1 which is a membrane for orbital fracturereconstruction as illustrated in FIG. 1, (6) and (6′) wherein the lengthm, width o and height c of the membrane are 30 to 50 mm, 20 to 40 mm and5 to 25 mm, respectively.
 9. A resorbable crosslinked form stablemembrane according to claim 8, wherein the thickness of the membrane is0.5 to 2.5 mm.
 10. A resorbable crosslinked form stable membraneaccording to claim 1, wherein the composite layer of collagen materialand inorganic ceramic particles contains 2.0 to 3.0 weight parts ofinorganic ceramic for 1 weight part of collagen material.
 11. Aresorbable crosslinked form stable membrane according to claim 1,wherein the collagen material comprises 70-90% collagen and 10-30%elastin.
 12. A resorbable crosslinked form stable membrane according toclaim 1, wherein the collagen material is derived from a porcine, bovineor equine peritoneum or pericardium membrane, small intestine mucosa(SIS) or muscle fascie.
 13. A resorbable crosslinked form stablemembrane according to claim 1, wherein one or both of the layers of theelastic pretensed collagen material includes holes of 5 to 1000 μm. 14.A resorbable crosslinked form stable membrane according to claim 1,wherein the inorganic mineral particles have a size of 150 to 500 μm.15. A resorbable crosslinked form stable membrane according to claim 1,wherein the inorganic ceramic is selected from the group consisting ofhydroxyapatite or hydroxyapatite bone mineral.
 16. A method ofregenerating human tissue outside of the oral cavity comprising applyingthe resorbable crosslinked form stable membrane of claim 1 to a surgicalsite in a human patient undergoing rhinoplasty, postlateral spinalfusion or orbital reconstruction.